Predicted structure of the adenovirus DNA binding protein

The DNA sequence of a portion of the MAV1SmaI-D fragment coding for the C-terminal 147 amino acids of the adenoviral DNA-binding protein (DBP) has been determined. A multiple sequence alignment was constructed of the MAV1 fragment and the DBPs of Ad.2, 4, 5, 7, 12, 40, and 41 to examine the degree of conservation of features that have been mapped on the Ad.2 DBP and to identify further conserved features. The less conserved N-terminal segment of the protein contains two nuclear localization signals and two acidic regions, the host range region, and all of the 11 phosphorylation sites. The highly conserved C-terminal segment contains a potential leucine zipper and zinc finger motifs. These sequence features were mapped onto a predicted secondary structure of the Ad.2 DBP.The nucleotide sequence data used in translation in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M81056     

Construction, characterization, and utilization of cell lines which inducibly express the adenovirus DNA-binding protein

To further our understanding of structure-function relationships within the multifunctional adenovirus DNA binding protein (DBP) a more diverse collection of mutants is necessary. DBP-expressing cell lines (gmDBP) were previously constructed that complemented DBP-negative mutants for viral growth. However, they did not allow severely defective viruses to form plaques. Since efficient mutant construction is reliant on plaque isolation of the desired mutant virus as a final step, additional gmDBP cell lines were constructed which allow all DBP-negative mutants to form plaques. Here we describe the construction and characterization of 12 new gmDBP cell lines. The utility of these lines was demonstrated by the efficient construction of a new defective mutant, H5in804, using a combination of DBP-expressing lines. The H5in804 mutation adds 22 amino acids at the carboxyl end of an otherwise wild type protein. Characterization of H5in804 revealed that it was altered in its ability to replicate viral DNA. The depression of DNA synthesis most probably results from a reduced ability of H5in804 DBP to bind ssDNA.     

诺如病毒衣壳蛋白重组人3型腺病毒的构建

目的 制备表达诺如病毒衣壳蛋白的重组人3型腺病毒.方法 将诺如病毒衣壳蛋白基因(Noro-orf2)克隆到腺病毒穿梭载体pBSE3CMV-egfp上,与线性化人3型腺病毒骨架质粒pBRAdv3共电转化感受态大肠杆菌BJ5183,使其在细菌内发生同源重组,带Noro-orf2基因的表达框置换腺病毒E3区,PCR及酶切筛选得到重组腺病毒质粒,将重组腺病毒质粒转染Hep-2细胞进行包装,获得感染性的重组腺病毒粒子,免疫组化分析重组腺病毒中诺如病毒衣壳蛋白的表达.结果 同源重组后经酶切和PCR鉴定证明插入Noro-orf2基因的重组腺病毒质粒pBRAdv3E3dNor成功构建,并经转染包装得到高滴度的重组腺病毒Adv3E3dNor,免疫组化证明诺如病毒衣壳蛋白得到表达.结论 成功构建表达诺如病毒衣壳蛋白的重组3型腺病毒Adv3E3dNor,为研制人3型腺病毒-诺如病毒双价疫苗奠定了基础.     

Comparative immunization in BALB/c mice with recombinant replication-defective adenovirus vector and DNA plasmid expressing a SARS-CoV nucleocapsid protein gene

In order to investigate immunogenicity in the induction of humoral and cellular immune responses, severe acute respiratory syndrome associated coronavirus （SARS-CoV）-N gene recombinant replication-defective adenoviral vector, rAd-N, was generated and immunized BALB/c mice in a pcDNA3.1-N prime-rAd-N boost regimen. After humoral and cellular immune response detection, different levels of SARS-CoV N protein specific antibodies and interferon-γ （IFN-γ） secretion are shown compared to controls. The humoral immune response was induced more effectively by the DNA priming and recombinant adenovirus boosting regimen. There is a significant difference between heterogeneous and homologous vaccinations. The heterogeneous combinations were all higher than those of the homologous combinations in the induction of anti-N antibody response. Among the three heterogeneous combinations, pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/rAd-N induced the strongest antibody response. In the induction of IFN-γ production, the homologous combination of rAd-N/rAd-N/rAd-N/rAd-N was significantly stronger than that of pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N/pcDNA3.1-N, but was relatively weaker than the heterogeneous combination of pcDAN3.1-N/pcDAN3.1-N/pcDAN3.1-N/rAd-N. This combination was a most efficient immunization regimen in induction of SARS-CoV-N-specific （IFN-γ） secretion just as the antibody response. These results suggest that DNA immunization followed by recombinant adenovirns boosting could be used as a potential SARS-CoV vaccine.     

Nbs1-dependent binding of Mre11 to adenovirus E4 mutant viral DNA is important for inhibiting DNA replication

Adenovirus (Ad) infections stimulate the activation of cellular DNA damage response and repair pathways. Ad early regulatory proteins prevent activation of DNA damage responses by targeting the MRN complex, composed of the Mre11, Rad50 and Nbs1 proteins, for relocalization and degradation. In the absence of these viral proteins, Mre11 colocalizes with viral DNA replication foci. Mre11 foci formation at DNA damage induced by ionizing radiation depends on the Nbs1 component of the MRN complex and is stabilized by the mediator of DNA damage checkpoint protein 1 (Mdc1). We find that Nbs1 is required for Mre11 localization at DNA replication foci in Ad E4 mutant infections. Mre11 is important for Mdc1 foci formation in infected cells, consistent with its role as a sensor of DNA damage. Chromatin immunoprecipitation assays indicate that both Mre11 and Mdc1 are physically bound to viral DNA, which could account for their localization in viral DNA containing foci. Efficient binding of Mre11 to E4 mutant DNA depends on the presence of Nbs1, and is correlated with a significant E4 mutant DNA replication defect. Our results are consistent with a model in which physical interaction of Mre11 with viral DNA is mediated by Nbs1, and interferes with viral DNA replication.     

腺病毒介导入B7—1基因对肝癌细胞免疫原性的影响

目的 研究同时含人ｐ５３、ＧＭ－ＣＳＦ、Ｂ７－１、ＩＬ－２基因的重组腺病毒载体Ａｄ－ｍｕｌｔｉｇｅｎｅｓ,对肝癌细胞免疫原性的影响。方法 应用人外周血淋巴细胞和肿瘤细胞的混合培养、外周血Ｔ淋巴细胞杀伤活性试验,分析导入目的基因的人肝癌细胞免疫原性的变化。结果 导入Ａｄ－ｍｕｌｔｉｇｅｎｅｓ的肝癌细胞系ＨｅｐＧ２、ＢＥＬ－７４０２,体外能刺激正常人外周血Ｔ淋巴细胞的增殖,并能增强Ｔ淋巴细胞的杀瘤细胞活性,加入ＩＬ－１２有协同增强作用。结论 肝癌细胞导入含Ｂ７－１基因的Ａｄ－ｍｕｌｔｉｇｅｎｅｓ后,其免疫原性得到增强。     

中国人IL—18结构蛋白cDNA的克隆和序列分析

目的克隆人白介素 - 18结合蛋白 (h IL- 18BP)的 c DNA,以研究其结构与功能的关系。方法从中国汉族人的胎盘组织中提取总 RNA,以 RT- PCR方法扩增出全长 5 85个 bp的 IL- 18BP c DNA,将其与表达载体 pc DNA3连接 ,转化大肠杆菌 DH5 α,建立了 h IL- 18BP的 c DNA克隆。结果序列分析表明 ,与国外文献报道的人 IL- 18BP的 c DNA序列完全一致。结论 h IL- 18BP已成功地得到克隆。     

重组腺病毒HIV-1 vpr基因的构建和表达

目的 构建携带HIV-1 vpr基因的重组腺病毒,使CD4 T淋巴细胞C8166内源性的高表达Vpr蛋白.方法 利用AdEasy-1系统, 通过将含有目的 基因片段的穿梭载体pAdTrack-CMV-vpr和骨架质粒pAdEasy-1在BJ5183细菌内同源重组的方法构建重组腺病毒质粒Ad-vpr, 用脂质体法将重组质粒转染至HEK293A细胞包装, 获得重组腺病毒Ad-vpr, 荧光显微镜观察Ad-vpr感染C8166细胞GFP的表达 , Western blotting鉴定Vpr在C8166细胞内的特异性表达,流式细胞术检测Ad-vpr感染 C8166细胞的效率.结果 成功构建携带HIV-1 vpr基因的重组腺病毒 ,Western blotting结果表明重组腺病毒Ad-vpr感染的C8166细胞内源性的高表达Vpr 蛋白,流式细胞术检测结果表明Ad-vpr感染C8166细胞效率高(44.07±3.62)%.结论 成功构建出携带HIV-1 vpr基因的重组腺病毒,使C8166细胞内源性的高表达Vpr蛋白.     

含E型沙眼衣原体MOMP基因的重组腺病毒和DNA疫苗联合免疫效果的研究

目的探讨E型沙眼衣原体（Ct）主要外膜蛋白（MOMP）DNA疫苗和重组腺病毒联合免疫小鼠诱导的免疫效应。方法构建、纯化重组腺病毒Ad-MOMP及重组真核表达质粒pVAX1-MOMP。设计4种免疫方案,分别为DNA免疫（ DNA组）、重组腺病毒免疫（ Ad组）、DNA初次免疫-重组腺病毒加强免疫（ DNA/Ad组）、重组腺病毒初次免疫-DNA加强免疫（ Ad/DNA组）。末次免疫后2周检测小鼠血清特异IgG、IgG1、IgG2a、IgA抗体,阴道分泌物SIgA抗体及脾淋巴细胞分泌IFN-γ、IL-10水平。结果DNA组诱导较弱免疫应答,未产生SIgA抗体及Th1反应。 Ad组诱导出Th1反应及SIgA抗体,且血清抗体显著高于DNA组。联合免疫均能诱导明显强于单独免疫的黏膜SIgA、血清抗体及Th1反应。 Ad/DNA组的Th1反应强于DNA/Ad组;而DNA/Ad组的血清抗体和黏膜抗体水平强于Ad/DNA组。结论Ad-MOMP能诱导黏膜免疫及Th1细胞免疫应答,DNA/Ad及Ad/DNA联合免疫产生的特异性免疫应答明显强于单独免疫。其中Ad/DNA的Th1反应优势更明显,DNA/Ad的血清抗体和黏膜抗体反应更强。接种顺序会影响联合免疫的强弱及类型,这为Ct疫苗的设计研究提供新的思路和实验依据。     

人胸腺基质淋巴生成素基因重组腺病毒载体的构建及表达

目的: 构建含人胸腺基质淋巴生成素基因(TSLP)的重组腺病毒载体并表达, 以研究其免疫学功能。方法: 将由人胚肺细胞扩增得到的TSLP基因, 克隆于真核表达载体pcDNA3. 1中, 再亚克隆至穿梭质粒pShuttle中, 并与腺病毒骨架载体pAdEasy -1共同转化大肠杆菌。以获得的重组质粒线性化后转染HEK293细胞, 并包装成病毒颗粒。采用PCR法对重组腺病毒基因进行鉴定, 并以Westernblot检测TSLP蛋白的表达。结果: 通过细菌内同源重组, 成功构建带有人胸腺基质淋巴生成素基因的重组腺病毒质粒, 转染 293细胞后, 包装的重组病毒经PCR检测表明, 基因组含有目的基因,病毒的滴度可达 1×1011pfu/L。Westernblot证实, 感染的肿瘤细胞中有相应基因产物的表达。结论: 通过菌内重组可高效制备带有特定基因的重组病毒。所制备的Ad -TSLP可成功表达相应基因产物, 为进一步研究这一新型细胞因子的功能奠定了基础。     

腺病毒转染法构建表达乙型肝炎病毒核心蛋白小鼠肝癌细胞系及其在乙肝疫苗评价中的应用

目的 构建一种可高效表达乙型肝炎病毒(Hepatitis B virus,HBV)核心蛋白(HBc)C57BL/6小鼠来源肝癌细胞系,并以其作为靶细胞评价乙肝基因疫苗在C57BL/6小鼠体内所引起的HBc特异性细胞毒性T细胞(CTL)的活性.方法 以HBV全基因组为模板,采用PCR方法扩增HBc基因,插入AdEasy腺病毒穿梭载体,构建携带HBc蛋白的腺病毒穿梭载体AD-HBc,电转携带有腺病毒骨架质粒(Ad-Easy)的E.coli BJ5183感受态细胞,获得重组腺病毒质粒AD-CMV-HBc,经PmeⅠ线性化处理后转染HEK293细胞进行包装得到相关的重组腺病毒,再感染C57BL/6小鼠来源肝癌细胞系Hepa1-6.结果 成功构建表达HBc蛋白的重组腺病毒,在体外对Hepa1-6细胞的感染率几乎为100％,并且HBc蛋白得到高效表达.以此为靶细胞用于pVAX1-HBc基因疫苗免疫C57BL/6小鼠产生的CTL活性的体外检测.结论 我们成功构建HBc蛋白表达的靶细胞系,对研究乙肝病毒核心抗原(HBcAg)所引起的细胞免疫反应及乙肝发病机理等方面有重要意义.     

1人TLR4胞外区基因重组腺病毒载体的构建与鉴定

目的 构建并鉴定人TLR4胞外区基因重组腺病毒载体.方法 以pUCm-TLR4质粒为模板,运用PCR技术扩增TLR4胞外区目的 基因片段,片段回收后经酶切连接穿梭质粒pAdTrack-CMV上,获得重组腺病毒质粒pAdTrack-CMV-TLR4.通过Kpn Ⅰ和HindⅢ双酶切,测序鉴定后,将鉴定正确的质粒经PmeⅠ酶切线性化后,转化到含有腺病毒骨架质粒pAdEasy-1的感受态细菌BJ5183中,进行同源重组,卡那抗性筛选阳性克隆,提取质粒,用脂质体介导转染293细胞,通过观察绿色荧光蛋白(GFP)的表达及PCR技术,Western blot等方法鉴定重组腺病毒,并进行病毒滴度测定.结果 人TLR4胞外区基因重组腺病毒载体构建正确,病毒滴度为3.2×109pfu/mL.结论 成功构建了人TLR4胞外区基因重组腺病毒载体,为下一步实验奠定基础.     

Karyotypic analysis of two related cervical carcinoma cell lines that contain human papillomavirus type 18 DNA and express divergent differentiation

The cell lines C-4I and C-4II were established in culture from a nonkeratinizing squamous carcinoma of the uterine cervix. Both lines contain human papillomavirus (HPV) type 18 DNA (Brandt et al., Cold Spring Harbor Laboratories, 5:179, 1987) and both are hypodiploid with similar, but not identical, karyotypes. Each line expresses multiple characteristics of ectocervical epithelial differentiation, but the characteristics differ between the lines. In the present study, G banding of the lines showed that cells of both lines have two normal chromosomes 1-5, 8-10, 13, 16, and 17, one normal chromosome 12 and 14, and no normal chromosomes 15 and 18. The lines share three abnormal chromosomes, der(8)t(8q;12q), der(18)t(18q;?), and i(5p). There are specific differences between the lines. C-4I has two normal chromosomes 6, while C-4II has one; C-4II has two chromosomes 11 and der(18)t(18q;?), while C-4I lacks both chromosomes 11 and has one der(18)t(18q;?). Each line has unique markers that include del(11)(p11), del(22)(q12), and del(21)(q21) in C-4I and i(15q), der(X)t(Xq;9p), der(6)t(6p;14q), and del(4)(q21) in C-4II. The results show that these phenotypically distinct lines are derived from the same clone and that the 8q arm (the site of HPV 18 integration) is present in three copies in both lines. They also define several chromosome rearrangements that are compatible with the expression of specific differentiation markers.     

反义人钙周期素结合蛋白编码基因转染对胃癌细胞多药耐药性的影响

目的 研究hCacyBP编码基因在胃癌多药耐药机制中的作用。方法 采用Northern杂交，检测SGC7901细胞及SGC7901／ADR细胞中hCacyBP mRNA表达水平的差异。将hCacyBP cDNA克隆到pcDNA3.1中，构建反义核酸真核表达载体pcDNA3.1/hCacyBP-，并转染耐阿霉素人胃癌细胞，用RT－PCR检测转染细胞中hCacyBP mRNA水平的变化。用MTT比色法和FCM，分别检测SGC7901／ADR细胞，pcDNA3.1/hCacyBP-和pcDNA3.1分别转染的SGC7901／ADR细胞，对ADR的药物敏感性和胞内ADR的蓄积浓度。结果 Northern杂交证实，SGC7901／ADR细胞中hCacyBP mRNA表达的水平显著高于SGC7901细胞。成功地构建了pcDNA3.1/hCacyBP-。以pcDNA3.1/hCacyBP-转染的SGC791／ADR细胞中hCacyBP mRNA的表达水平，显著低于空载体转染及未转染的SGC791／ADR细胞。MTT检测表明，转染pcDNA3.1/hCacyBP-细胞，对ADR的药物敏性较空载体转染及未转染的SGC7901／ADR细胞有所增高，生存率较后两者为低。FCM显示，转染pcDNA3.1/hCacyBP-的SGC7901／ADR细胞，转染pcDNA3.1及未转染的SGC7901／ADR细胞内ADR的蓄积浓度，分别为6．72，5．62和5．54。结论 hCacyBP编码基因对胃癌细胞的MDR有一定的影响，CacyBP可能是一种胃癌细胞MDR中的重要分子。     

Crystal structure of TtgV in complex with its DNA operator reveals a general model for cooperative DNA binding of tetrameric gene regulators

The majority of bacterial gene regulators bind as symmetric dimers to palindromic DNA operators of 12–20 base pairs (bp). Multimeric forms of proteins, including tetramers, are able to recognize longer operator sequences in a cooperative manner, although how this is achieved is not well understood due to the lack of complete structural information. Models, instead of structures, of complete tetrameric assembly on DNA exist in literature. Here we present the crystal structures of the multidrug-binding protein TtgV, a gene repressor that controls efflux pumps, alone and in complex with a 42-bp DNA operator containing two TtgV recognition sites at 2.9 Å and 3.4 Å resolution. These structures represent the first full-length functional tetrameric protein in complex with its intact DNA operator containing two continuous recognition sites. TtgV binds to its DNA operator as a highly asymmetric tetramer and induces considerable distortions in the DNA, resulting in a 60° bend. Upon binding to its operator, TtgV undergoes large conformational changes at the monomeric, dimeric, and tetrameric levels. The structures here reveal a general model for cooperative DNA binding of tetrameric gene regulators and provide a structural basis for a large body of biochemical data and a reinterpretation of previous models for tetrameric gene regulators derived from partial structural data.     

血管抑素K1-5腺病毒载体的构建及对血管内皮细胞的抑制作用

目的构建携带血管抑素的腺病毒载体,并探讨其对血管内皮细胞增殖及迁移的抑制作用。方法采用基因重组技术,构建携带血管抑素K1-5的腺病毒载体,PCR鉴定,50%组织培养感染剂量法测定病毒滴度及氯化铯密度梯度离心法纯化病毒。直接感染台盼蓝染色排除法、MTT及体外趋化实验检测血管抑素K1-5对内皮细胞增殖及趋化的抑制作用。结果经50%组织培养感染剂量法测定病毒滴度及氯化铯密度梯度离心法纯化病毒,病毒滴度为1.5×109pfu/mL。扩增纯化后,病毒滴度达1.1×1010pfu/mL。实验表明,携带血管抑素K1-5的腺病毒能抑制血管内皮细胞增生,并对其具有杀伤力,且能使血管内皮细胞趋化能力减弱,抑制血管形成。结论成功构建了携带血管抑素K1-5的腺病毒载体,血管抑素K1-5能明显抑制血管内皮细胞增殖及迁移,为进一步体内实验,研究对血管形成的抑制作用奠定了基础。     

Autoimmunity to a 28-30 kD cell membrane DNA binding protein: occurrence in selected sera from patients with SLE and mixed connective tissue disease (MCTD).

Previous experiments have established the presence of a 30-kD DNA binding protein on the surface of human leukocytes. Herein we report that selected sera from patients with systemic lupus erythematosus (SLE) and MCTD are reactive with a 28-30 kD protein on immunoblots of peripheral blood mononuclear cells (PBMC) cell membrane preparations; the reactivity is abolished by prior incubation of the blot with DNA. Antibodies eluted from the 28-30 kD strip inhibited the binding of 3H. DNA to human PBMC. An immunomatrix of 28-30 kD reactive immunoglobulins was able to extract a 29-kD DNA binding protein from a PBMC cell membrane preparation. Flow cytometry experiments confirmed the cell surface IgG reactivity of sera with T lymphocytes. Additional experiments indicated that cell surface IgG binding was not due to antibodies binding to cell surface DNA, DNA anti-DNA immune complexes reacting with a DNA binding protein, anti-histone antibodies or anti-Sm antibodies. It is hypothesized that this autoimmune response could be one component of an idiotypic network involving anti-DNA antibodies.